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We narrowed to 594 results for: cel.2

Showing: 541 - 560 of 594 results

  1. Immunocytochemistry

    Type
    Protocol
    ...HeLa cells per well. Allow the HeLa cells to grow to the desired density before labeling. Section 2: Fixing...coated coverslips HeLa cells 24-well plate 4% Paraformaldehyde 5 mg/mL 4′,6-diamidino-2-phenylindole (DAPI...Fluorescent microscope 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel...of the diluted antibody to the wells and incubate 2 h at RT . Remove the primary antibody and dispose ...detect antigens in cells. Here we describe the basic steps for fixing and labeling cells in culture with ...2022 Workflow Timeline Day 1: Seed cells Day 3-4: Fix and label cells Equipment Pipette controller Pipette... Seeding cells Place a sterile poly-D-lysine coated coverslip in each well of a 24-well cell culture treated...

  2. Bioinformatics at Addgene

    Type
    Blog Post
    ...the necessary steps to perform:      Fig. 2: Quick-stepping to a FASTA file   With our outline...mission and our own, as both address the need to accelerate science using different approaches. We were also...evolved into a single, organized pipeline and accelerated the processing of our sequencing data. However...

  5. Getting the Most from Your Lentiviral Transduction

    Type
    Blog Post
    ...infection of cultured cells.” Nature 339 (1989). 487–488. PubMed PMID: 2725683. 2. Miller, C.J., Kassem...Love your cells and they'll love you back A successful lentiviral infection begins with your cells, both ...both your packaging cells and your target cells. Poor tissue culture practices can lead to low titers and... promote cell growth. Overgrown cultures stimulate quiescence, the resting phase of the cell cycle; quiescent...quiescent cells do not take up nucleic acid or express transgenes as well as actively dividing cells resulting...reaching 108 cells per ml before the media becomes cloudy. Mycoplasma compete with host cells for nutrients... your producer cells with fresh medium. Daily medium exchanges help alleviate cellular stresses associated...

  6. Proximity Labeling: A Powerful Tool for Protein Complex Purification and Proteomic Mapping

    Type
    Blog Post
    ...approximately 2-4 million proteins per cubic micron in bacteria, yeast, and mammalian cells (Milo, 2013)...plasmids for RapID EXCELL: Proximity labeling for the detection of cell-cell interactions EXCELL is an enzyme-mediated...enzyme-mediated proximity cell labeling strategy to detect cell-cell interactions in vivo (Ge et al., 2019...et al. "Enzyme-Mediated Intercellular Proximity Labeling for Detecting Cell–Cell Interactions." Journal...a bait protein fused to BirA* in cells and then incubate those cells with exogenous biotin. After a few...peptide (LPETG). In EXCELL, the mgSrtA variant can be displayed on the surface of a cell of interest, where...-protein interactions, and even more recently cell-cell interactions. If you’re looking to identify interactions...

  7. CRISPR Library Amplification

    Type
    Protocol
    ...times are limited. Day 2: Harvest cells and purify DNA (Estimated time 3-4 hours) Cells should be harvested... recover, set up overnight growth (Estimated time 2-3 hours) Transformation should be performed at the... Tubes should contain a total of 5 mL (3 mL SOC + 2 mL transformed Endura from two separate transformations...has been absorbed by the agar. This usually takes 1-2 minutes. Critical Be careful not to rip or shred the...incubation if needed. Place 100 mL sterile LB at 4 ℃. Day 2 (morning) Before beginning, prechill at least four...pellet. The total weight of each pellet should be ~1-2 g. Pro-Tip Make sure to weigh the empty tube beforehand...electrocompetent cells (Default: 4 tubes of Endura Duos, Lucigen, 60242-1) Alternatives include Stbl4 cells or other...

  8. Four Base Editing Reporters to Monitor and Enrich Editing in Real-time

    Type
    Blog Post
    ...positive cells by the number of only mCherry positive cells (Martin et al., 2019). Figure 2: Editing...restoring mCherry expression. Cells can then be sorted using Fluorescence-activated cell sorting (FACS) and the...easily determined by comparing cells expressing only eGFP expression to cells expressing eGFP and mCherry...expressed in cells. mCherry is constitutively expressed to identify successfully transfected cells. C-to-T ...TREE) to purify single base edited cells without the need for single cell isolation and downstream sequencing...fluorescence and cell sorting using FACS which could limit their use across different cell types and systems...sgRNA targeting the GFPACG to the cells. As expected, only the cells that contained the cytidine base ...

  9. The Fluorescent Vegetables in Aptamer Soup

    Type
    Blog Post
    ...of 3,5-difluoro-4-hydroxybenzylidene-imidazolinone-2-oxime (DFHO), a fluorophore modified from DsRed. DFHO...challenging to track intracellularly, but aptamers permit the study of intracellular RNA dynamics. Aptamers...RNA aptamers are used to track cellular processes by transforming cells with plasmids encoding aptamer-tagged... been used to investigate intracellular viral DNA dynamics in neuronal cells (Nilaratanakul et al., 2017...be used to simultaneously target cellular RNA to specific intracellular locations using Cas9 and track ...and later introducing the small molecule into the cell. The fluorescence that is generated allows for qualitative...). DFHBI is membrane permeable and non toxic to cells. Because it rapidly photobleaches, DFHBI can only...

  10. Adeno-associated Viruses (AAVs) for Genome Editing

    Type
    Blog Post
    ...PMCID: PMC3010411. 2. Kohli, Manu, et al. "Facile methods for generating human somatic cell gene knockouts...K562 leukemia cell line and ~8 and 9% efficiencies in T-cells and hematopoietic stem cells respectively...transient transfection of 293T cells with helpers, and used to infect recipient cells. After G418 selection, ...using AAV vectors for gene editing in mammalian cells since 1998. In that year, Russell and Hirata from...used to routinely deliver sequences of interest to cells with efficiencies of 1-40% after G418 selection ...the p53 and PTEN tumor suppressor genes in human cells (Kim et al 2008). They have also used AAV gene editing...endogenous allele of the STAG2 tumor suppressor in human cells (Kim et al, 2016). The Waldman lab has designed ...

  11. Plasmids 101: E. coli Strains for Protein Expression

    Type
    Blog Post
    ...gene1 [lon] ompT gal sulA11 R(mcr-73::miniTn10--TetS)2 [dcm] R(zgb-210::Tn10--TetS) General protein expression... want to make sure your cells have first reached an appropriate density. Cells in the exponential growth...collecting dead cells, and, conversely, you cannot induce too early as there are not enough cells in the culture...host protein, using up valuable resources in the cell thus leading to slowed growth. Additionally, some...such as TB or 2XYT is optimal because of the high cell-density they support; however, minimal media supplemented...

  12. Protocol - Over-Agar Antibiotic Plating

    Type
    Protocol
    ...or other cell spreading device that fits the plate. Incubate plates at 37 ℃ for 18 hours. Day 2 Observe...individual colonies and effective selection. 150 µL of 2 mg/mL Carbenicillin plated over-agar Plate shows less...individual colonies with smaller size than the 1 mg/mL and 2 mg/mL plates and effective selection. Selection Curve...investigator to conveniently plate and select transformed cells containing plasmids differing in their resistance...bent to create an “L” shape, and then used like a cell spreader. Several other devices may be used for.... During the incubation, transform DH5α E. coli cells by heatshock with the plasmid of interest. See our...the liquid is mostly absorbed. The spreading of cells can be done in the same way as the antibiotic, using...

  14. 5 Reasons to Use Reddit for Science Communication

    Type
    Blog Post
    ...community is active, engaged, and has great questions! 2. The r/Science subreddit is well moderated. One of...questions from the AMA - as you’ll see below, it pretty nicely displays her energetic, truthful and helpful style... training is abundant, helpful, and sometimes excellent. Finally, as I said in the AMA, it’s incredibly...topics to the Addgene blog The blog has many other excellent posts on science career topics as well. Instead...

  15. Viral Vectors 101: Systemic Capsids

    Type
    Blog Post
    ...start selecting and testing systemic capsids! Figure 2 contains a decision tree for selecting a capsid, while... highlighted below the table.     Figure 2: This decision tree can help guide your choice on ...transfer to the adult brain. Nature Biotechnology, 34(2), 204–209. https://doi.org/10.1038/nbt.3440 Goertsen... your cell type of interest, while miRNA target sites can be used to prevent expression in cell types ...endothelial cells in rodent CNS AAV-PHP.V1 shows higher transduction in CNS endothelial cells (60%) and ...endothelial cells in the retina and spinal cord (Krolak et al., 2022). Systemic capsids for broad cell type ...mouse to primate neocortex. Cell Reports, 34(13). https://doi.org/10.1016/j.celrep.2021.108754 Ravindra Kumar...

  16. Targeting HIV-1 with CRISPR: Shock and Kill or Cut it Out?

    Type
    Blog Post
    ... genome from every infected cell and 2. prevent reinfection of those cells. Kaminski et al. designed gRNAs...infected cells, the other using wild-type Cas9 to remove the HIV-1 genome from infected cells. Read on...1 reservoirs HIV-1 infects cells in the immune system, notably CD4+ T-cells, and eventually leads to acquired...percentage of cells with activated HIV-1, measured via HIV-1 promoter-driven GFP expression. In cell lines that...Kaminski et al. observed no negative effects on cell viability, cell cycle progression, or apoptosis with Cas9...in HIV-1 infected T-cells to see if they could be rescued by CRISPR. CD4+ T-cells were isolated from healthy...1 is limited to a subset of T-cells, but it is unclear if enough T-cells could be reached to ablate a ...

  17. Five Popular Model Organisms

    Type
    Blog Post
    ...variety of environmental conditions, and double every 2 hours. Yeast are also the first eukaryotic genome ... properties and processes with our own cells. Like human cells, yeast DNA is packaged into chromosomes...differentiation of β-cells in the the developing zebrafish pancreas. They first tagged these cells using plasmids...recapitulate the complex interactions between cancer cells, therapeutic drugs, and the rest of the body than...than studies in other model organisms or cell culture. For example, Scott Lowe’s lab utilized mice to study...to identify a set of mutations that made cancer cells more resistant to chemotherapy. You can find these...to optimally control gene expression in specific cells of the fly (Pfeiffer et al., 2010). Yeast (Saccharomyces...

  18. Hot Biosensors 2022: Year-End Roundup

    Type
    Blog Post
    ... more from the authors in this blog post. Figure 2: iGluSnFR3 displays enhanced brightness, expression...two-photon microscopy. Cell. 2022 Aug 16. pii: S0092-8674(22)00916-3. doi: 10.1016/j.cell.2022.07.013 pYtags...Calcium Indicator. Front Cell Dev Biol. 2022 Jun 29;10:880107. doi: 10.3389/fcell.2022.880107. KRaION potassium...fluorescence unit fused with TRansmembrane region-to-IntrAcellular loop linkers” (MTRIA for short), where a fluorescent...inserting a circularly-permuted GFP into an intracellular loop of various G-protein-coupled receptors....fluorescent sensor for real-time measurement of extracellular oxytocin dynamics in the brain. Nat Methods....sensor relies on a change in localization to the cell membrane and not a direct change in the fluorescent...

  19. Quick Guide to Near-Infrared Fluorescent Proteins

    Type
    Blog Post
    ...Oligomeric state Photo-stabilityc, t1/2, s pKa Brightness in HeLa cells vs. iRFP713, % d Ref BphP-based...mammalian cells. dDetermined as effective NIR fluorescence in transiently transfected live HeLa cells with ...Near-Infrared Fluorescent Proteins in Mammalian Cells. Cell Chem Biol 24, 758-766 e753 (2017). Baloban, M...developers and users, BV is present in eukaryotic cells as an intermediate of heme metabolism (Shemetov ...to GFP-like probes by supplying a single gene to cells. Still in bacteria, which do not produce BV, an ... to consider effective brightness of NIR FPs in cells, in addition to their molecular brightness (a product...the concentration of BV-containing molecules in a cell and depends on the efficiency of BV incorporation...

  20. Fluorescent Proteins 101: GFP Fusion Proteins - Making the Right Connection

    Type
    Blog Post
    ..., whereas the mVenus-APT1 fusion does not (figure 2). In the mVenus-APT1, a lipidation motif, which is...proteins enable the observation of proteins in living cells and organisms. Both components of the chimera are...However, these may not reflect the situation in cells. Therefore, the localization of critical fusions...currently the standard for assessing oligomerization in cells. In addition to measuring stickiness, several researchers...fluorescent proteins that behave as monomers in mammalian cells include: mTurquoise2, mEGFP, mNeonGreen, mScarlet...protein to the existing pool of protein within a cell and may lead to over-expression artifacts. This ...heterotrimeric G protein Gq activation in living cells." BMC biology 9.1 (2011): 32. PubMed PMID: 21619590...
Showing: 541 - 560 of 594 results